εndotoxin

Before endoscopy, blood was drawn from a peripheral vein to measure endotoxin and cytokines in all participants. Circulating endotoxin concentration in serum samples was determined via ELISA using commercially available kits, as per the manufacturer’s instructions (Εndotoxin cat#abx051541; Abbexa Ltd., Cambridge Science Park, Cambridge, UK, range = 0.015–2 EU/mL, sensitivity < 0.005), and systemic inflammatory response was assessed by determining cytokine levels (IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α) using flow cytometry. Cytokine concentration in serum samples was measured with a BD FACS Array Bioanalyzer, using a cytometric bead array (CBA) assay (Human Inflammatory Cytokines Kit, cat#551811, BD Biosciences) (range 20–5000 pg/mL, sensitivity 7.2 pg/mL for IL-1β, 2.5 pg/mL for IL-6, 3.6 pg/mL for IL-8, 3.3 pg/mL for IL-10, 1.9 pg/mL for IL-12, and 3.7 pg/mL for TNF-α). The cytokine concentrations were measured using a Cytometric Bead Array methodology that combines the principles of ELISA and flow cytometry to quantify soluble proteins. As such, protein concentrations that fall below the standard curve of the assay cannot be safely quantified. The samples that fell below range were measured without dilution to ensure the accuracy of the results and subsequently reported as “0”.

Xanthine oxidase (XOD), myeloperoxidase (MPO), and zonulin plasma levels were measured using ELISA kits (Cusabio^® Technology Llc, Houston, TX, USA). Resolvin D1 (RvD1) levels were measured in plasma using an ELISA kit (Cayman Chemical^®, Ann Arbor, MI, USA). Cytokeratin 18 (CK-18) levels were measured also in plasma using an M30 Apoptoense^® ELISA kit (VLVbio AB, Nacka, Sweden) in which the units measured are defined against a native antigen, and it is calibrated against a recombinant protein standard that 1 U/L = 1.24 pM. Ectodysplasin-A (EDA) plasma levels were determined using an ELISA kit (Assay Genie, Dublin, Ireland). Chemerin and omentin levels were measured in plasma and in serum respectively using ELISA kits (Abcam^®, The Netherlands, Amsterdam). IL-1β, IL-1ra, IL-6, MCP-1, TNFα and leptin levels were determined in plasma using Human Custom ProcartaPlex^TM (Invitrogen by Thermo Fisher Scientific, Bender MedSystems GmbH, Vienna, Austria). The concentrations of endotoxin were measured in plasma by a commercially available kit Εndotoxin (Abbexa Ltd., Cambridge Science Park, Cambridge, UK). All ELISA kits were carried out following the supplier’s guidelines for use.